EGCG、Taurine和Genistein聯(lián)合抗大鼠肝纖維化作用的關鍵蛋白驗證研究
發(fā)布時間：2018-08-19 14:08
【摘要】：目的:以表沒食子兒茶素沒食子酸酯(EGCG)、牛磺酸(Taurine)和三羥基異黃酮(Genistein)三種具有不同抗纖維化作用的藥物組成聯(lián)合藥物干預CC14誘導的肝纖維化大鼠及肝星狀細胞HSC-T6后,對前期用i TRAQ標記聯(lián)合質(zhì)譜的技術篩選和鑒定出肝星狀細胞、大鼠肝組織及血清中差異表達的9個蛋白質(zhì)Mst4、CDK4、Fgb、Tpi1、FVII、Hdgf、Glud 1、Lamp1、Cathepsin D進行深入的體內(nèi)外驗證;并從中選出感興趣的蛋白FVII,構建穩(wěn)定慢病毒載體對HSC-T6中FVII進行基因干擾,篩選到穩(wěn)轉株后,采用聯(lián)合藥物干預,觀察細胞行為學變化,以對FVII蛋白在肝纖維化發(fā)生發(fā)展中的功能進行更深一步的研究。研究結果以期驗證聯(lián)合用藥抗肝纖維化作用的關鍵蛋白,探討聯(lián)合用藥抗肝纖維化作用的機理,為肝纖維化的藥物篩選及早期診斷提供幫助。方法:1.關鍵蛋白的體內(nèi)外驗證研究:(1)以CC14誘導大鼠肝纖維化模型,在造模成功后給予EGCG、Taurine和Genistein組成的聯(lián)合藥物治療6周,采集大鼠血清和肝組織,HE染色觀察肝組織病理變化。(2)以HSC-T6細胞株為對象,應用聯(lián)合藥物對其進行干預后收集細胞。(3)提取肝組織及細胞的RNA和蛋白質(zhì),采用Western blot和RT-qPCR技術分別在mRNA和蛋白水平對9個可能的關鍵蛋白進行驗證,并用ELISA法測定血清中FVII的含量。2.關鍵蛋白FVII的功能分析:(1)運用RNAi技術構建并篩選出穩(wěn)定過表達FVII的慢病毒載體,構建FVII基因過表達的穩(wěn)轉細胞株。(2)CCK-8法檢測過表達FVII的HSC-T6增殖情況,分析FVII對HSC-T6增殖的影響。(3)聯(lián)合藥物干預過表達FVII的HSC-T6,CCK-8法檢測各組細胞的藥物抑制率,分析fvii過表達后對聯(lián)合藥物干預hsc-t6的增殖影響。(4)流式細胞術檢測聯(lián)合藥物干預的hsc-t6親本株、fvii過表達hsc-t6細胞株的細胞周期變化,分析fvii過表達后對聯(lián)合藥物干預hsc-t6的細胞周期的影響。結果:1.關鍵蛋白的體內(nèi)外驗證結果:(1)給藥六周后,he染色結果顯示,聯(lián)合用藥組能減輕大鼠纖維化程度。(2)westernblot檢測發(fā)現(xiàn),聯(lián)合用藥干預后,與模型組相比,大鼠肝組織fⅦ、cdk4、hdgf蛋白表達量顯著降低;與對照組相比,藥物組肝星狀細胞中l(wèi)amp1、mst4、fⅦ蛋白表達量降低,tpi1蛋白表達量升高,差異有統(tǒng)計學意義。(3)real-timeqpcr檢測發(fā)現(xiàn),與模型組相比,聯(lián)合用藥組大鼠肝組織中l(wèi)amp1、fvii、hdgf、glud1的表達量降低,差異有統(tǒng)計學意義。與對照組相比,聯(lián)合用藥組細胞中fvii的mrna表達水平在用藥后降低,fgb、tpi1的mrna表達量升高,差異有統(tǒng)計學意義。(4)elisa結果顯示,與模型組相比,藥物干預6周后聯(lián)合用藥組大鼠血清中fvii含量降低,差異有統(tǒng)計學意義。2.關鍵蛋白fvii的功能分析結果:(1)cck-8法繪制hsc-t6親本株、nc株及fⅦ過表達株細胞的生長曲線,發(fā)現(xiàn)fⅦ過表達后,肝星狀細胞增殖加快。(2)在fⅦ過表達hsc-t6細胞株中,高劑量藥物對細胞增殖抑制顯著,低、中劑量組藥物對細胞增殖抑制不顯著;在hsc-t6親本株與nc株中,中、高劑量藥物對細胞抑制顯著;低劑量藥物對細胞抑制不顯著,說明fⅦ的過表達,阻礙了聯(lián)合用藥抑制hsc-t6的增殖。進一步分析發(fā)現(xiàn),相同濃度的聯(lián)合藥物對hsc-t6株及nc株的抑制率高于lv-fⅦ株,且中劑量藥物組差異有統(tǒng)計學意義(p0.01),從而表明在聯(lián)合用藥抗肝纖維化作用中,fⅦ可能降低了聯(lián)合用藥抗肝纖維化的效果。(3)流式細胞儀檢測發(fā)現(xiàn),不同濃度藥物處理hsc-t6親本株24h后,細胞的周期發(fā)生了變化,與對照組相比,隨著藥物濃度增加,s期百分比升高,g1期百分比下降,而g2期百分比沒有顯著改變;中濃度藥物組與高濃度藥物組細胞與對照組相比,s期及G1期百分比變化有統(tǒng)計學差異,P0.05,低濃度藥物組與對照組相比細胞周期各期百分比無明顯改變。應用不同濃度藥物處理過表達株LV-FⅦ24h后,細胞周期發(fā)生了變化,與對照組相比,隨著藥物濃度增加,S期百分比升高,G2期百分比均下降,G1期百分比變化沒有顯著差異;中濃度藥物組與高濃度藥物組細胞與對照組相比,S期及G2期百分比變化有統(tǒng)計學差異,P0.05,低濃度藥物組與對照組相比細胞周期各期百分比無明顯改變,表明聯(lián)合藥物處理后肝星狀細胞增殖被抑制,細胞周期被阻滯于S期。結論:1.應用Western blot和RT-qPCR技術分別在細胞和動物水平對聯(lián)合用藥抗肝纖維化作用的9個候選關鍵蛋白進行了mRNA和蛋白驗證,其中Fgb、Tpi1、CDK4、Mst4、FⅦ的基因和蛋白差異表達水平與前期的研究結果一致,可能為聯(lián)合用藥抗肝纖維化作用的關鍵蛋白。我們推測聯(lián)合用藥可能是通過調(diào)控Fgb、Tpi1、CDK4、Mst4、FⅦ等關鍵蛋白的表達,從而影響溶酶體、糖酵解、抑制DNA復制和有絲分裂、抗氧化防御系統(tǒng)以及凝血級聯(lián)反應等多重細胞信號途徑起到抗纖維化作用。2.依據(jù)驗證結果選擇FⅦ進一步對其在聯(lián)合用藥抗肝纖維化作用的功能進行分析后,發(fā)現(xiàn)FVII過表達后,肝星狀細胞增殖被促進。過表達FⅦ會降低肝星狀細胞對聯(lián)合用藥的敏感性,能減輕藥物對肝星狀細胞的抑制,推測可能FⅦ起到藥效的負調(diào)控的作用。聯(lián)合藥物干預24h后,親本株及FⅦ過表達株的細胞周期都受到了抑制,推斷聯(lián)合用藥可能是通過將細胞周期阻滯于S期,抑制HSC的增殖。相同濃度的藥物對FⅦ過表達前后的HSC-T6細胞周期的干預沒有明顯差異,說明FⅦ可能不是通過調(diào)控細胞周期來影響細胞對藥物的反應,具體機制還需深入探索。
[Abstract]:OBJECTIVE: To screen and identify hepatic stellate cells (HSC-T6) and CC14-induced hepatic fibrosis in rats with hepatic stellate cells (HSC-T6) treated by epigallocatechin gallate (EGCG), taurine (Taurine) and trihydroxy isoflavone (Genistein). Differentially expressed 9 proteins Mst4, CDK4, Fgb, Tpi1, FVII, Hdgf, Glud 1, Lamp1, Cathepsin D in stellate cells, rat liver tissues and serum were further validated in vitro and in vivo, and interesting proteins FVII were selected from them to construct stable lentiviral vector to interfere with FVII gene in HSC-T6. To observe the changes of cellular behavior and further study the function of FVII protein in the occurrence and development of hepatic fibrosis. The results were expected to verify the key proteins of anti-hepatic fibrosis effect of combination therapy, and to explore the mechanism of anti-hepatic fibrosis effect of combination therapy, so as to provide help for drug screening and early diagnosis of hepatic fibrosis. In vitro and in vivo validation of the key proteins: (1) CC14-induced rat liver fibrosis model, after successful modeling, the rats were treated with EGCG, Taurine and Genistein for 6 weeks. Serum and liver tissues were collected, and the pathological changes of liver tissues were observed by HE staining. (2) HSC-T6 cell line was used as the object, and the combined drugs were used to interfere with it. Collector cells. (3) RNA and protein were extracted from liver tissues and cells. Nine possible key proteins were identified at mRNA and protein levels by Western blot and RT-q PCR. Serum FVII levels were determined by ELISA. Functional analysis of key protein FVII was carried out. (1) Slow over-expression of FVII was constructed and screened by RNAi technique. (2) The proliferation of HSC-T6 overexpressing FVII was detected by CCK-8 method, and the effect of FVII on the proliferation of HSC-T6 was analyzed. (3) The drug inhibition rate of HSC-T6 overexpressing FVII was detected by CCK-8 method, and the effect of FVII overexpressing on the proliferation of HSC-T6 was analyzed. 4) Flow cytometry was used to detect t he cell cycle changes of HSC-T6 parental strains which were interfered with drugs. The effect of FVII overexpression on t he cell cycle of HSC-T6 was analyzed. Results: 1. The results of in vitro and in vivo validation of t he key proteins: (1) After six weeks of administration, HE staining showed that t he combination group could significantly reduce t he cell cycle. (2) Western blot showed that the expression of f_, cdk4, HDGF protein in rat liver tissue was significantly lower than that in model group after treatment, while the expression of lamp1, mst4, f_protein in hepatic stellate cells was significantly lower and the expression of tpi1 protein was significantly higher in drug group than that in control group. (3) Real-time qPCR assay Compared with the model group, the expression of lamp-1, fvii, hdgf, glud-1 in the liver tissue of rats in the combined treatment group was significantly lower than that in the control group. compared with the control group, the expression of FVII mRNA in the cells of the combined treatment group was decreased, and the expression of fgb, tpi-1 mRNA was increased, the difference was statistically significant. (4) ELISA results showed that compared with the model, the expression of FVII mRNA in the cells of the combined treatment group was decreased. Compared with the control group, the content of FVII in the serum of rats in the combined treatment group decreased after 6 weeks of drug intervention, and the difference was statistically significant. 2. functional analysis of the key protein fvii: (1) the growth curve of HSC-T6 parental strain, NC strain and f_overexpression strain was drawn by CCK-8 method, and it was found that the proliferation of hepatic stellate cells was accelerated after f_overexpression. (2) the fine expression of HSC-T6 in f_was observed. In cell lines, high-dose drugs inhibited cell proliferation significantly, while low-dose drugs inhibited cell proliferation insignificantly; in HSC-T6 parental and NC strains, high-dose drugs inhibited cell proliferation significantly; low-dose drugs inhibited cell proliferation insignificantly, suggesting that the over-expression of f_inhibited the proliferation of hsc-t6. It was found that the inhibitory rate of the same concentration of combination drugs on HSC-T6 and NC strains was higher than that of lv-f_strains, and the difference was statistically significant in the middle dose group (p0.01). Thus, in the anti-hepatic fibrosis effect of combination drugs, f_may reduce the anti-hepatic fibrosis effect of combination drugs. (3) Flow cytometry detection found that different concentrations of drug treatment. Compared with the control group, with the increase of drug concentration, the percentage of S phase increased, the percentage of G1 phase decreased, but the percentage of G2 phase did not change significantly. Compared with the control group, the percentage of S phase and G1 phase changed significantly in the middle concentration group and the high concentration group, P 0.05, the percentage of low concentration group. Compared with the control group, the percentage of S phase increased, that of G2 phase decreased, and that of G1 phase did not change significantly. Compared with the control group, the percentage changes of S and G2 phases were statistically significant. P 0.05, there was no significant change in the percentage of cell cycle in the low concentration group compared with the control group, indicating that the proliferation of hepatic stellate cells was inhibited and the cell cycle was blocked in S phase after treatment with combined drugs. CR technique was used to verify the mRNA and protein expression of nine candidate key proteins of anti-hepatic fibrosis effect of combination therapy at the cellular and animal levels. The differential expression levels of Fgb, Tpi1, CDK4, Mst4 and F genes and proteins were consistent with previous studies, which may be the key proteins of anti-hepatic fibrosis effect of combination therapy. The combination of Fgb, Tpi1, CDK4, Mst4, F may play an anti-fibrosis role by regulating the expression of key proteins such as Fgb, Tpi1, CDK4, Mst4, F, thus affecting lysosome, glycolysis, inhibiting DNA replication and mitosis, antioxidant defense system and coagulation cascade reaction. After analyzing the function of anti-hepatic fibrosis, it was found that the proliferation of hepatic stellate cells was promoted after overexpression of FVII. Overexpression of F could reduce the sensitivity of hepatic stellate cells to combination therapy, and alleviate the inhibition of drug on hepatic stellate cells. It was speculated that F might play a negative role in regulating the efficacy of the drug. The cell cycle of HSC-T6 overexpressed strains was inhibited, and it was concluded that the combination therapy might inhibit the proliferation of HSC by blocking the cell cycle at S phase. The specific mechanism needs further exploration.
【學位授予單位】：廣西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R575.2
本文編號：2191886
[Abstract]:OBJECTIVE: To screen and identify hepatic stellate cells (HSC-T6) and CC14-induced hepatic fibrosis in rats with hepatic stellate cells (HSC-T6) treated by epigallocatechin gallate (EGCG), taurine (Taurine) and trihydroxy isoflavone (Genistein). Differentially expressed 9 proteins Mst4, CDK4, Fgb, Tpi1, FVII, Hdgf, Glud 1, Lamp1, Cathepsin D in stellate cells, rat liver tissues and serum were further validated in vitro and in vivo, and interesting proteins FVII were selected from them to construct stable lentiviral vector to interfere with FVII gene in HSC-T6. To observe the changes of cellular behavior and further study the function of FVII protein in the occurrence and development of hepatic fibrosis. The results were expected to verify the key proteins of anti-hepatic fibrosis effect of combination therapy, and to explore the mechanism of anti-hepatic fibrosis effect of combination therapy, so as to provide help for drug screening and early diagnosis of hepatic fibrosis. In vitro and in vivo validation of the key proteins: (1) CC14-induced rat liver fibrosis model, after successful modeling, the rats were treated with EGCG, Taurine and Genistein for 6 weeks. Serum and liver tissues were collected, and the pathological changes of liver tissues were observed by HE staining. (2) HSC-T6 cell line was used as the object, and the combined drugs were used to interfere with it. Collector cells. (3) RNA and protein were extracted from liver tissues and cells. Nine possible key proteins were identified at mRNA and protein levels by Western blot and RT-q PCR. Serum FVII levels were determined by ELISA. Functional analysis of key protein FVII was carried out. (1) Slow over-expression of FVII was constructed and screened by RNAi technique. (2) The proliferation of HSC-T6 overexpressing FVII was detected by CCK-8 method, and the effect of FVII on the proliferation of HSC-T6 was analyzed. (3) The drug inhibition rate of HSC-T6 overexpressing FVII was detected by CCK-8 method, and the effect of FVII overexpressing on the proliferation of HSC-T6 was analyzed. 4) Flow cytometry was used to detect t he cell cycle changes of HSC-T6 parental strains which were interfered with drugs. The effect of FVII overexpression on t he cell cycle of HSC-T6 was analyzed. Results: 1. The results of in vitro and in vivo validation of t he key proteins: (1) After six weeks of administration, HE staining showed that t he combination group could significantly reduce t he cell cycle. (2) Western blot showed that the expression of f_, cdk4, HDGF protein in rat liver tissue was significantly lower than that in model group after treatment, while the expression of lamp1, mst4, f_protein in hepatic stellate cells was significantly lower and the expression of tpi1 protein was significantly higher in drug group than that in control group. (3) Real-time qPCR assay Compared with the model group, the expression of lamp-1, fvii, hdgf, glud-1 in the liver tissue of rats in the combined treatment group was significantly lower than that in the control group. compared with the control group, the expression of FVII mRNA in the cells of the combined treatment group was decreased, and the expression of fgb, tpi-1 mRNA was increased, the difference was statistically significant. (4) ELISA results showed that compared with the model, the expression of FVII mRNA in the cells of the combined treatment group was decreased. Compared with the control group, the content of FVII in the serum of rats in the combined treatment group decreased after 6 weeks of drug intervention, and the difference was statistically significant. 2. functional analysis of the key protein fvii: (1) the growth curve of HSC-T6 parental strain, NC strain and f_overexpression strain was drawn by CCK-8 method, and it was found that the proliferation of hepatic stellate cells was accelerated after f_overexpression. (2) the fine expression of HSC-T6 in f_was observed. In cell lines, high-dose drugs inhibited cell proliferation significantly, while low-dose drugs inhibited cell proliferation insignificantly; in HSC-T6 parental and NC strains, high-dose drugs inhibited cell proliferation significantly; low-dose drugs inhibited cell proliferation insignificantly, suggesting that the over-expression of f_inhibited the proliferation of hsc-t6. It was found that the inhibitory rate of the same concentration of combination drugs on HSC-T6 and NC strains was higher than that of lv-f_strains, and the difference was statistically significant in the middle dose group (p0.01). Thus, in the anti-hepatic fibrosis effect of combination drugs, f_may reduce the anti-hepatic fibrosis effect of combination drugs. (3) Flow cytometry detection found that different concentrations of drug treatment. Compared with the control group, with the increase of drug concentration, the percentage of S phase increased, the percentage of G1 phase decreased, but the percentage of G2 phase did not change significantly. Compared with the control group, the percentage of S phase and G1 phase changed significantly in the middle concentration group and the high concentration group, P 0.05, the percentage of low concentration group. Compared with the control group, the percentage of S phase increased, that of G2 phase decreased, and that of G1 phase did not change significantly. Compared with the control group, the percentage changes of S and G2 phases were statistically significant. P 0.05, there was no significant change in the percentage of cell cycle in the low concentration group compared with the control group, indicating that the proliferation of hepatic stellate cells was inhibited and the cell cycle was blocked in S phase after treatment with combined drugs. CR technique was used to verify the mRNA and protein expression of nine candidate key proteins of anti-hepatic fibrosis effect of combination therapy at the cellular and animal levels. The differential expression levels of Fgb, Tpi1, CDK4, Mst4 and F genes and proteins were consistent with previous studies, which may be the key proteins of anti-hepatic fibrosis effect of combination therapy. The combination of Fgb, Tpi1, CDK4, Mst4, F may play an anti-fibrosis role by regulating the expression of key proteins such as Fgb, Tpi1, CDK4, Mst4, F, thus affecting lysosome, glycolysis, inhibiting DNA replication and mitosis, antioxidant defense system and coagulation cascade reaction. After analyzing the function of anti-hepatic fibrosis, it was found that the proliferation of hepatic stellate cells was promoted after overexpression of FVII. Overexpression of F could reduce the sensitivity of hepatic stellate cells to combination therapy, and alleviate the inhibition of drug on hepatic stellate cells. It was speculated that F might play a negative role in regulating the efficacy of the drug. The cell cycle of HSC-T6 overexpressed strains was inhibited, and it was concluded that the combination therapy might inhibit the proliferation of HSC by blocking the cell cycle at S phase. The specific mechanism needs further exploration.
【學位授予單位】：廣西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R575.2
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